DNA polymerases catalyze DNA polymerization. In addition, a subset of Family A, B, and D DNA polymerases also have proofreading 3′ to 5′ (3′-5′) exonuclease activity and are referred to as exo+ polymerases (Blanco et al. Gene 100: 27-38 (1991)). When a DNA polymerase incorporates an incorrect or modified nucleotide, for example, in a primer strand, it detects structural perturbations caused by mispairing or nucleotide modification and transfers the primer strand from the polymerase domain to the 3′-5′ exonuclease active site.
These polymerases have been extensively employed in molecular biology applications such as single-molecule sequencing, sequencing by synthesis, and single nucleotide polymorphism (SNP) detection. Modified nucleotides that may be incorporated by DNA polymerases in these methods include nucleotide terminators (dideoxynucleotide triphosphates (ddNTPs), and acyclic-nucleoside triphosphates (acycloNTPs)), reversible nucleotide terminators (3′-O-azidomethyl-ddNTPs, 3′-O-amino-ddNTPs, and Lightning Terminators™ (Lasergen, Inc., Houston, Tex.)) and tagged nucleotides (biotin-deoxyuridine triphosphates (biotin-dUTPs)). Once incorporated, these modified nucleotides can be hydrolyzed by DNA polymerases having exonuclease activity, compromising the incorporation regimen.
Presumptive exonuclease minus (exo−) DNA polymerase mutants have been described in the literature and are commercially available. The commercial exo− archaeal DNA polymerase mutants have a single mutation in Motif I and/or II, or a double mutation in Motif I, namely D141A and E143A, that reportedly abolishes detectible exonuclease activity (see for example, VENT® (Thermococcus litoralis) (Kong et al. J. Biol. Chem. 268(3):1965-1975) (New England Biolabs, Inc. (NEB), Ipswich, Mass.); Thermococcus JDF-3 (U.S. Pat. No. 6,946,273, U.S. 2005-0069908); KODI (Thermococcus kodakaraensis) (U.S. Pat. No. 6,008,025); Pfu (Pyrococcus furiosus) (U.S. Pat. No. 5,489,523, U.S. Pat. No. 7,704,712, and U.S. Pat. No. 7,659,100); and 9° N (Thermococcus sp.) (U.S. 2005-0123940 and Southworth et al. Proc Natl Acad Sci USA 93:5281-5285 (1996)).